Sites Of Tissue Injury Research Articles

Pharmacological antagonism of Ccr2+ cell recruitment to facilitate regenerative tendon healing.

Successful tendon healing requires sufficient deposition and remodeling of new extracellular matrix at the site of injury, with this process mediating in part through fibroblast activation via communication with macrophages. Moreover, resolution of healing requires clearance or reversion of activated cells, with chronic interactions with persistent macrophages impairing resolution and facilitating the conversion to fibrotic healing. As such, modulation of the macrophage environment represents an important translational target to improve the tendon healing process. Circulating monocytes are recruited to sites of tissue injury, including the tendon, via upregulation of cytokines including Ccl2, which facilitates recruitment of Ccr2+ macrophages to the healing tendon. Our prior work has demonstrated that Ccr2-/- can modulate fibroblast activation and myofibroblast differentiation. However, this approach lacked temporal control and resulted in healing impairments. Thus, in the current study we have leveraged a Ccr2 antagonist to blunt macrophage recruitment to the healing tendon in a time-dependent manner. We first tested the effects of Ccr2 antagonism during the acute inflammatory phase and found that this had no effect on the healing process. In contrast, Ccr2 antagonism during the early proliferative/granulation tissue period resulted in significant improvements in mechanical properties of the healing tendon. Collectively, these data demonstrate the temporally distinct impacts of modulating Ccr2+ cell recruitment and Ccr2 antagonism during tendon healing and highlight the translational potential of transient Ccr2 antagonism to improve the tendon healing process.

Tinea capitis with kerion complicated by keloid formation in a 34-year-old man: A case report

A kerion, also known as a dermatophyte, is a type of inflammatory tinea capitis caused by an exaggerated immune response to a fungal ringworm infection primarily affecting the scalp. A keloid is an abnormal scar tissue proliferation occurring at the site of tissue injury or spontaneously. It occurs more commonly in darker-skinned individuals. The coexistence of kerion and keloid is rare. This case involves a 34-year-old man who presented with a four-year history of scalp swelling, itching, and occasional fluid discharge. There is a history of keloids in multiple parts of the body. Examination revealed multiple keloids on the scalp alongside a purulent, oozing rash with satellite nodules. Skin scraping for KOH microscopy was positive for fungal spores and hyphae. This case highlights the rare complication of keloid formation in kerion which can hinder effective treatment of the underlying fungal infection.

Pharmacological Antagonism of Ccr2+ Cell Recruitment to Facilitate Regenerative Tendon Healing.

Successful tendon healing requires sufficient deposition and remodeling of new extracellular matrix at the site of injury, with this process mediating in part through fibroblast activation via communication with macrophages. Moreover, resolution of healing requires clearance or reversion of activated cells, with chronic interactions with persistent macrophages impairing resolution and facilitating the conversion the conversion to fibrotic healing. As such, modulation of the macrophage environment represents an important translational target to improve the tendon healing process. Circulating monocytes are recruited to sites of tissue injury, including the tendon, via upregulation of cytokines including Ccl2, which facilitates recruitment of Ccr2+ macrophages to the healing tendon. Our prior work has demonstrated that Ccr2-/- can modulate fibroblast activation and myofibroblast differentiation. However, this approach lacked temporal control and resulted in healing impairments. Thus, in the current study we have leveraged a Ccr2 antagonist to blunt macrophage recruitment to the healing tendon in a time-dependent manner. We first tested the effects of Ccr2 antagonism during the acute inflammatory phase and found that this had no effect on the healing process. In contrast, Ccr2 antagonism during the late inflammatory/ early proliferative period resulted in significant improvements in mechanical properties of the healing tendon. Collectively, these data demonstrate the temporally distinct impacts of modulating Ccr2+ cell recruitment and Ccr2 antagonism during tendon healing and highlight the translational potential of transient Ccr2 antagonism to improve the tendon healing process.

INHIBITION OF INTEGRIN VLA-3 AND TETRASPANIN CD151 PROTECTS AGAINST NEUTROPHIL-MEDIATED ENDOTHELIAL DAMAGE.

Background: The recruitment of neutrophils to sites of localized injury or infection is initiated by changes on the surface of endothelial cells located in proximity to tissue damage. Inflammatory mediators, such as TNF-α, increase surface expression of adhesive ligands and receptors on the endothelial surface to which neutrophils tether and adhere. Neutrophils then transit through the activated endothelium to reach sites of tissue injury with little lasting vascular injury. However, in cases of sepsis, the interaction of endothelial cells with highly activated neutrophils can cause damage vascular damage. The identification of molecules that are essential for neutrophil diapedesis may reveal targets of therapeutic opportunity for preservation of endothelial function in the presence of critical illness. We tested the hypothesis that inhibition of neutrophil β1 integrin very late antigen-3 (VLA-3; α3β1) and/or inhibition of the tetraspanin (TM4) family member CD151 would protect against neutrophil-mediated loss of endothelial function. Methods: Blood was obtained from septic patients or healthy donors. Neutrophils were purified, and aliquots were treated with/without proinflammatory molecules. Confluent human umbilical vascular endothelial cells were activated with TNF-α. Electric cell impedance sensing was used to determine monolayer resistance over time after the addition of neutrophils that were treated with blocking antibodies against VLA-3 and/or CD151 or isotype controls. Groups (depending on relevancy) were analyzed by Mann-Whitney U test, Wilcoxon test, or repeated-measures one-way ANOVA. Results: Neutrophils from septic patients and neutrophils activated ex vivo reduced endothelial monolayer resistance to a greater extent than neutrophils from healthy donors. Antibody blockade of VLA-3 and/or CD151 significantly reduced activation-associated endothelial damage. Similar findings were demonstrated on fibronectin, collagen I, collagen IV, and laminin, suggesting that neutrophil surface VLA-3 and CD151 are responsible for endothelial damage regardless of substrata and are likely to be operative in all bodily tissues. Conclusion: This report identifies VLA-3 and CD151 on the activated human neutrophil, which are responsible for damage to endothelial function. Targeting these molecules in vivo may demonstrate preservation of organ function during critical illness.

A nox2/cybb zebrafish mutant with defective myeloid cell reactive oxygen species production displays normal initial neutrophil recruitment to sterile tail injuries.

Reactive oxygen species are important effectors and modifiers of the acute inflammatory response, recruiting phagocytes including neutrophils to sites of tissue injury. In turn, phagocytes such as neutrophils are both consumers and producers of reactive oxygen species. Phagocytes including neutrophils generate reactive oxygen species in an oxidative burst through the activity of a multimeric phagocytic nicotinamide adenine dinucleotide phosphate oxidase complex. Mutations in the NOX2/CYBB (previously gp91phox) nicotinamide adenine dinucleotide phosphate oxidase subunit are the commonest cause of chronic granulomatous disease, a disease characterized by infection susceptibility and an inflammatory phenotype. To model chronic granulomatous disease, we made a nox2/cybb zebrafish (Danio rerio) mutant and demonstrated it to have severely impaired myeloid cell reactive oxygen species production. Reduced early survival of nox2 mutant embryos indicated an essential requirement for nox2 during early development. In nox2/cybb zebrafish mutants, the dynamics of initial neutrophil recruitment to both mild and severe surgical tailfin wounds was normal, suggesting that excessive neutrophil recruitment at the initiation of inflammation is not the primary cause of the "sterile" inflammatory phenotype of chronic granulomatous disease patients. This nox2 zebrafish mutant adds to existing in vivo models for studying reactive oxygen species function in myeloid cells including neutrophils in development and disease.

Abstract 1130: Tenascin-C In Arteriovenous Fistula Failure: Unraveling Venous Remodeling Dynamics

Background: AVF are the preferred vascular access for hemodialysis, with superior patency and lower infection rates than grafts or catheters. Nevertheless, AVF patency is reduced by both early thrombosis and NIH, culminating in diminished utilization due to one-year patency rates as low as 40-50%. Tenascin-C (TnC), an ECM glycoprotein with limited adult expression, is transiently present at sites of tissue injury and inflammation. TnC has been implicated in playing a role in the development of NIH. Hypothesis: Sustained TnC expression contributes to NIH, potentially leading to AVF failure. Methods: Female and male wild-type C57Bl/6 mice, aged 9-11 weeks, had sham surgery or AVF creation by puncturing the infrarenal aorta into the inferior vena cava (IVC) with a 25-gauge needle. Doppler ultrasound confirmed AVF presence. Tissue samples were collected for IF, WB, and qPCR. Human matched vein and AVF tissue were obtained from patients undergoing planned two-stage AVF creation surgeries and analyzed with IF. In vitro analysis of human dermal microvascular endothelial cells (HDMEC) was performed to investigate the role of TnC in thrombomodulin expression. Results: TnC expression significantly increased in the AVF by day 7, returning to baseline by day 21 and reaching negligible levels by day 42. The spatial and temporal regulation of TnC expression post-AVF creation correlated with ECM remodeling and inversely with thrombomodulin. Both mice and human studies demonstrated differential regulation of TnC and thrombomodulin in patent and occluded AVF. In HDMEC, TnC knockdown suppressed NF-kB expression and increased thrombomodulin expression, while exogenous TnC had the opposite effect. Conclusion: TnC plays a pivotal role in driving pathological inflammation, with its expression being both temporally and spatially regulated during venous remodeling post-AVF creation. Persistent TnC expression is noted in occluded mouse and human AVF, leading to decreased thrombomodulin expression and heightened thrombogenicity. Sustained TnC expression regulates the shift from physiological to pathological venous remodeling, underscoring the crucial role of the immune response in this process and highlighting potential translational therapeutic strategies.

The Effect of a Rotating Magnetic Field on the Regenerative Potential of Platelets.

Platelets are actively involved in tissue injury site regeneration by producing a wide spectrum of platelet-derived growth factors such as PDGF (platelet-derived growth factor), IGF-1 (insulin-like growth factor), TGF-β1 (transforming growth factor β), FGF (fibroblast growth factor), etc. A rotating magnetic field (RMF) can regulate biological functions, including reduction or induction regarding inflammatory processes, cell differentiation, and gene expression, to determine the effect of an RMF on the regenerative potential of platelets. The study group consisted of 30 healthy female and male volunteers (n = 15), from which plasma was collected. A portion of the plasma was extracted and treated as an internal control group. Subsequent doses of plasma were exposed to RMF at different frequencies (25 and 50 Hz) for 1 and 3 h. Then, the concentrations of growth factors (IGF-1, PDGF-BB, TGF-β1, and FGF-1) were determined in the obtained material by the ELISA method. There were statistically significant differences in the PDGF-BB, TGF-β1, IGF-1, and FGF-1 concentrations between the analyzed groups. The highest concentration of PDGF-BB was observed in the samples placed in RMF for 1 h at 25 Hz. For TGF-β1, the highest concentrations were obtained in the samples exposed to RMF for 3 h at 25 Hz and 1 h at 50 Hz. The highest concentrations of IGF-1 and FGF-1 were shown in plasma placed in RMF for 3 h at 25 Hz. An RMF may increase the regenerative potential of platelets. It was noted that female platelets may respond more strongly to RMF than male platelets.

Structural insights into the biological functions of the long pentraxin PTX3.

Soluble pattern recognition molecules (PRMs) are a heterogenous group of proteins that recognize pathogen- and danger-associated molecular patterns (PAMPs and DAMPs, respectively), and cooperate with cell-borne receptors in the orchestration of innate and adaptive immune responses to pathogenic insults and tissue damage. Amongst soluble PRMs, pentraxins are a family of highly conserved proteins with distinctive structural features. Originally identified in the early 1990s as an early inflammatory gene, PTX3 is the prototype of long pentraxins. Unlike the short pentraxin C reactive protein (CRP), whose expression is mostly confined to the liver, PTX3 is made by several immune and non-immune cells at sites of infection and inflammation, where it intercepts fundamental aspects of infection immunity, inflammation, and tissue remodeling. Of note, PTX3 cross talks to components of the complement system to control cancer-related inflammation and disposal of pathogens. Also, it is an essential component of inflammatory extracellular matrices (ECMs) through crosslinking of hyaluronic acid and turn-over of provisional fibrin networks that assemble at sites of tissue injury. This functional diversity is mediated by unique structural characteristics whose fine details have been unveiled only recently. Here, we revisit the structure/function relationships of this long pentraxin in light of the most recent advances in its structural biology, with a focus on the interplay with complement and the emerging roles as a component of the ECM. Differences to and similarities with the short pentraxins are highlighted and discussed.

A biofunctional review of C-reactive protein (CRP) as a mediator of inflammatory and immune responses: differentiating pentameric and modified CRP isoform effects.

C-reactive protein (CRP) is an acute phase, predominantly hepatically synthesized protein, secreted in response to cytokine signaling at sites of tissue injury or infection with the physiological function of acute pro-inflammatory response. Historically, CRP has been classified as a mediator of the innate immune system, acting as a pattern recognition receptor for phosphocholine-containing ligands. For decades, CRP was envisioned as a single, non-glycosylated, multi-subunit protein arranged non-covalently in cyclic symmetry around a central void. Over the past few years, however, CRP has been shown to exist in at least three distinct isoforms: 1.) a pentamer of five identical globular subunits (pCRP), 2.) a modified monomer (mCRP) resulting from a conformational change when subunits are dissociated from the pentamer, and 3.) a transitional isoform where the pentamer remains intact but is partially changed to express mCRP structural characteristics (referred to as pCRP* or mCRPm). The conversion of pCRP into mCRP can occur spontaneously and is observed under commonly used experimental conditions. In careful consideration of experimental design used in published reports of in vitro pro- and anti-inflammatory CRP bioactivities, we herein provide an interpretation of how distinctive CRP isoforms may have affected reported results. We argue that pro-inflammatory amplification mechanisms are consistent with the biofunction of mCRP, while weak anti-inflammatory mechanisms are consistent with pCRP. The interplay of each CRP isoform with specific immune cells (platelets, neutrophils, monocytes, endothelial cells, natural killer cells) and mechanisms of the innate immune system (complement), as well as differences in mCRP and pCRP ligand recognition and effector functions are discussed. This review will serve as a revised understanding of the structure-function relationship between CRP isoforms as related to inflammation and innate immunity mechanisms.

#6679 ANALYSIS OF MACROPHAGE POPULATIONS IN THE KIDNEY WITH RENAL TUBULAR CELL-SPECIFIC SENESCENCE INDUCTION

Abstract Background and Aims Ageing is a risk factor for multiple diseases, including kidney disease. To improve quality of life, pharmaceutical treatments are given to ageing patients, often with side-effects and cross-reactions. Research is needed into the effects and mechanisms of ageing in order to identify new druggable targets to improve quality of life. Cells can become senescent with age and are seen at sites of tissue injury. Senescent cells (SCs) undergo irreversible cell-cycle arrest, but remain metabolically active, producing a range of signalling molecules called the senescence associated secretory phenotype (SASP, made up of pro-inflammatory/fibrotic elements). Senescent cells within the kidney post-acute kidney injury (AKI) may be involved in ongoing damage, leading in some cases to chronic kidney disease (CKD), possibly due to interactions with macrophages. We hypothesise that the recruitment of monocytes/macrophages by senescent cells is due to the signalling components present in their SASP. Method Pax8 is renal tubule marker. We use Pax8-creERT2;mdm2 fl/fl mice to study kidney-specific senescence induction in absence of age/injury. Upon administration of tamoxifen, the mdm2 alleles are floxxed out by cre recombinase, allowing p53 to stabilise and activate the p21CIP1 cell-cycle inhibitor, inducing senescence in Pax8+ kidney-specific cells. The use of an endogenous tdTomato reporter allows visualisation of cells undergoing successful recombination upon the administration of tamoxifen (TAM). Results As expected, cell-cycle inhibitor/SC marker, Cdkn1a was upregulated by qPCR in TAM-treated young murine whole kidneys 10-fold compared to controls (p = 0.0173) (Figure 1). Ccl2 (monocyte chemoattractant) was up-regulated 20-fold in young (p = 0.0405) TAM-treated TG mice vs controls (Figure 1). In agreement with an increase of Ccl2 transcripts, there was a significant increase of renal macrophages as quantified by flow cytometry of kidney digests (p = 0.0141) in TAM treated young TG mice. Immunofluorescence staining of transgenic mice revealed an increase of Iba1 positive macrophages, correlated with staining of tdTomato tubules, suggesting that increase in macrophage numbers was in response to senescence induction (Figure 2). Iba1-p21 co-stains show a increases in p21 positive cells in TAM-treated transgenic mice, evidently a result of construct activation and appear in proximity to the macrophages (Figure 2). Conclusion Apparent increases in macrophages due to the presence of senescent cells may indicate one of the mechanisms by which AKI progresses to CKD, particularly if inflammatory macrophage phenotypes are present and persist. Macrophage and monocyte populations fluctuate with age and the use of this model allows analysis of macrophage recruitment/polarisation due to senescent cell burden in the absence of age/injury and possible confounding factors, in young mice.

The RNA editor ADAR2 promotes immune cell trafficking by enhancing endothelial responses to interleukin-6 during sterile inflammation

Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to this response remains elusive. We report that RNA editor ADAR2 exerts a tissue- and stress-specific regulation of endothelial responses to interleukin-6 (IL-6), which tightly controls leukocyte trafficking in IL-6-inflamed and ischemic tissues. Genetic ablation of ADAR2 from vascular endothelial cells diminished myeloid cell rolling and adhesion on vascular walls and reduced immune cell infiltration within ischemic tissues. ADAR2 was required in the endothelium for the expression of the IL-6 receptor subunit, IL-6 signal transducer (IL6ST; gp130), and subsequently, for IL-6 trans-signaling responses. ADAR2-induced adenosine-to-inosine RNA editing suppressed the Drosha-dependent primary microRNA processing, thereby overwriting the default endothelial transcriptional program to safeguard gp130 expression. This work demonstrates a role for ADAR2 epitranscriptional activity as a checkpoint in IL-6 trans-signaling and immune cell trafficking to sites of tissue injury.

Surface-Functionalized Stem Cell-Derived Extracellular Vesicles for Vascular Elastic Matrix Regenerative Repair.

Extracellular vesicles (EVs) are nanosized vesicles that carry cell-specific biomolecular information. Our previous studies showed that adult human bone marrow mesenchymal stem cell (BM-MSC)-derived EVs provide antiproteolytic and proregenerative effects in cultures of smooth muscle cells (SMCs) derived from an elastase-infused rat abdominal aortic aneurysm (AAA) model, and this is promising toward their use as a therapeutic platform for naturally irreversible elastic matrix aberrations in the aortic wall. Since systemically administered EVs poorly home into sites of tissue injury, disease strategies to improve their affinity toward target tissues are of great significance for EV-based treatment strategies. Toward this goal, in this work, we developed a postisolation surface modification strategy to target MSC-derived EVs to the AAA wall. The EVs were surface-conjugated with a short, synthetic, azide-modified peptide sequence for targeted binding to cathepsin K (CatK), a cysteine protease overexpressed in the AAA wall. Conjugation was performed using a copper-free click chemistry method. We determined that such conjugation improved EV uptake into cultured aneurysmal SMCs in culture and their binding to the wall of matrix injured vessels ex vivo. The proregenerative and antiproteolytic effects of MSC-EVs on cultured rat aneurysmal SMCs were also unaffected following peptide conjugation. From this study, it appears that modification with short synthetic peptide sequences seems to be an effective strategy for improving the cell-specific uptake of EVs and may be effective in facilitating AAA-targeted therapy.

Osteochondral Tissue Engineering Dilemma: Scaffolding Trends in Regenerative Medicine.

Orthopedic surgeons face a lot of difficulties in managing and repairing osteochondral defects. Damaged articular cartilage and the subchondral bone underneath are both present in osteochondral defects. The demands of the bone, cartilage, and the contact between the bone and the cartilage must be taken into consideration while repairing an osteochondral defect. Only palliative, not curative, therapeutic interventions are now available for the healing of osteochondral abnormalities. With its ability to successfully rebuild bone, cartilage, and the junction between bone and cartilage, tissue engineering has been recognized as an effective substitute. In correlation, mechanical stress and physical processes are commonly applied to the osteochondral area. Therefore, the ability of chondrocytes and osteoblasts to regenerate is influenced by bioactive molecules and the physicochemical characteristics of the surrounding matrix. The treatment of osteochondral disorders is said to benefit from the use of stem cells as an alternative intervention. In the field of tissue engineering, various approaches have been used such as the direct implantation of scaffolding materials at the site of tissue injury in patients, either alone or loaded with cells and bioactive molecules at the target site to imitate the natural extracellular matrix. Despite the extensive use and advancements of tissue-engineered biomaterials such as natural and synthetic polymer-based scaffolds, their repair capacity is limited due to challenges in combating antigenicity, designed to simulate in vivo microenvironment, and conducting mechanical or metabolic characteristics comparable to native organs/tissues. This study explores numerous osteochondral tissue engineering methodologies focusing on scaffold design, material varieties, manufacturing techniques, and functional features. This review is focused on recent breakthroughs in bioactive scaffolds that aid osteogenic and chondrogenic differentiation for bone and cartilage repair. The topic will cover fundamental anatomy, osteochondral repair methodologies and obstacles, cell selection, biochemical variables, and bioactive materials, as well as the design and manufacture of bioactive scaffolds. Additionally, we focus on the concept and construction of decellularized scaffolds, and the fabrication of dECM scaffolds in tissue engineering from various skin, bone, nerve, heart tissue, lung, liver, and kidney, and their application in osteochondral regeneration.

Abstract 639: Prevention of pancreatitis and subsequent pancreatic cancer and fibrosis development by PI3Kγ inhibition

Abstract Anti-inflammatory, immunosuppressive macrophages play significant roles in promoting both tumorigenesis and fibrosis. Wound-healing type macrophages accumulate in sites of tissue injury, such as the cirrhotic liver, where they can induce oxidative damage and secrete immunosuppressive and pro-fibrotic factors that lead to oncogenesis. These innate immune cells are also major cellular producers of PDGFB and TGF-β1, which stimulate fibroblasts to secrete extracellular matrix proteins, such as collagen, and which also promote metastatic progression of neoplastic diseases. Chronic liver and pancreatic inflammation are significant risk factors for the development of hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma (PDAC), respectively. We have determined that the myeloid cell specific PI3K isoform PI3Kγ promotes the accumulation of myeloid cells and their immune suppressive polarization within the tumor microenvironment (TME), and that this kinase thereby promotes tumor growth in animal models of cancer, including PDAC. Genetic and pharmacological inhibition of PI3Kγ suppresses tumor growth and reduces fibrosis that is associated with PDAC and other tumors. Targeting immune suppressive myeloid cells represent an emerging approach within the field of cancer immunotherapy and fibrosis treatment; indeed, inhibition of PI3Kγ by the therapeutic eganelisib has shown beneficial effects, including increased overall and progression-free survival in clinical trials for melanoma, head and neck carcinoma, urothelial carcinoma and triple negative breast carcinoma. In this project, we are exploringWe examined the effect of whether PI3Kγ inhibition can in cancer prevention using mouse models of prevent cancer development by suppressing damaging chronic hepatic and pancreatic inflammation, associated fibrosis, and progression towards tumorigenesis using models of HCC and PDAC. We found that PI3Kγ inhibition prevented prevents acute and chronic inflammation and that promotes normal tissue loss and development of fibrosis in mouse models of chronic pancreatitis and liver inflammation. Studies are underway to determine iOur studies indicate thatf PI3Kγ inhibition can also prevent the development of pancreatic and liver carcinoma that results fromin response to chronic inflammation and fibrosis. Our These studies thus far suggest the a potential role of for PI3Kγ inhibition in the prevention of cancer development by preventing the chronic inflammation that leads towards fibrosis and carcinogenesis. The results of these studies could be applied to the future design of clinical trials with using clinical PI3Kγ inhibitors. Citation Format: Anghesom Ghebremedhin, Judith Varner, Marc Paradise. Prevention of pancreatitis and subsequent pancreatic cancer and fibrosis development by PI3Kγ inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 639.

Regenerative capacity of trophoblast stem cell-derived extracellular vesicles on mesenchymal stem cells

BackgroundHuman mesenchymal stem cells (MSCs) are therapeutic for clinical applications because of their excellent immunomodulatory and multiple lineage differentiation abilities at tissue injury sites. However, insufficient number of cells and lack of regenerative properties during in vitro expansion still limit the clinical applicability of MSC therapies. Here, we demonstrated a preconditioning strategy with trophoblast stem cell-derived extracellular vesicles (TSC-EVs) to boost the proliferation and regenerative capacity of MSCs.MethodsWe employed cell proliferation analyses such as CCK8 and BrdU assays to determine the proliferation-promoting role of TSC-EVs on MSCs. Osteogenic effects of TSC-EVs on MSCs were assessed by alkaline phosphatase (ALP) activity, calcium assays, and calvarial bone defect animal models. For skin regenerative effects, skin wound mice model was exploited to analyze wound-healing rate in this study, as well as immunofluorescence and histological staining evaluates. We also performed the small RNA profiling and RNA-sequencing analyzes to understand the cellular mechanism of TSC-EVs on MSCs.ResultsTSC-EVs significantly promoted MSC proliferation under xeno-free conditions and facilitated the therapeutic effects of MSCs, including osteogenesis, anti-senescence, and wound healing. Transcriptomic analysis also provided evidence that specific microRNAs in TSC-EVs and differentially expressed genes (DEGs) in TSC-EV-treated MSCs showed the possibility of TSC-EVs triggering the regenerative abilities of MSCs with cytokine interaction. Hence, we found that NGF/Akt signaling mediated the regenerative effects of TSC-EVs on MSCs as a particular cellular signaling pathway.ConclusionThe results of this study demonstrated the functional properties of TSC-EVs on MSCs for MSC-based therapeutic applications, suggesting that TSC-EVs may serve as a potential preconditioning source for MSC therapy in the clinical field of regenerative medicine.Graphical abstract

Stem cell- derived extracellular vesicles as new tools in regenerative medicine - Immunomodulatory role and future perspectives.

In the last few decades, the practical use of stem cells (SCs) in the clinic has attracted significant attention in the regenerative medicine due to the ability of these cells to proliferate and differentiate into other cell types. However, recent findings have demonstrated that the therapeutic capacity of SCs may also be mediated by their ability to secrete biologically active factors, including extracellular vesicles (EVs). Such submicron circular membrane-enveloped vesicles may be released from the cell surface and harbour bioactive cargo in the form of proteins, lipids, mRNA, miRNA, and other regulatory factors. Notably, growing evidence has indicated that EVs may transfer their bioactive content into recipient cells and greatly modulate their functional fate. Thus, they have been recently envisioned as a new class of paracrine factors in cell-to-cell communication. Importantly, EVs may modulate the activity of immune system, playing an important role in the regulation of inflammation, exhibiting broad spectrum of the immunomodulatory activity that promotes the transition from pro-inflammatory to pro-regenerative environment in the site of tissue injury. Consequently, growing interest is placed on attempts to utilize EVs in clinical applications of inflammatory-related dysfunctions as potential next-generation therapeutic factors, alternative to cell-based approaches. In this review we will discuss the current knowledge on the biological properties of SC-derived EVs, with special focus on their role in the regulation of inflammatory response. We will also address recent findings on the immunomodulatory and pro-regenerative activity of EVs in several disease models, including in vitro and in vivo preclinical, as well as clinical studies. Finally, we will highlight the current perspectives and future challenges of emerging EV-based therapeutic strategies of inflammation-related diseases treatment.

Autologous platelet-rich plasma in the delayed union of long bone fractures – A quasi experimental study

IntroductionFractures of long bones unite without any complication except for 2%–10% which may lead to delayed or non-union of the fracture. Management of delayed union of fractures poses a great challenge for orthopaedic surgeons. Platelet-rich plasma (PRP) is an autologous blood-derived biological agent, which delivers growth factors, cytokines, and bio-micro molecules at supraphysiologic concentrations at the site of tissue injury, thus potentiating the body's healing efforts. Various studies and research have proved the osteogenic activity of PRP. The growth factors present in the PRP induce the locally available resilient progenitor or stem cells and convert the atrophic environment into a trophic environment. Materials and methodsWe investigated the safety and efficacy of autologous PRP injection in the delayed union of long bone fractures. A total of 25 cases of delayed union of long bone fractures were augmented with 3 doses of autologous PRP at 3 weekly intervals and were followed up for 12 months. All the cases were documented with pre-and post-procedural and 12th -month visual analog score (VAS) and Warden's score. ResultsOut of 25 cases, 21 (84.00%) cases showed good union of fracture with adequate callus formation by 10–12 weeks with 3 doses of autologous PRP injections. The mean pre-procedural VAS and Warden's score at the final follow-up showed statistically significant results (p < 0.05). No other complications were noted due to autologous PRP application among the study participants during the study period except for 3 cases (2 cases of non-union, and 1 case of implant failure). ConclusionResults of the current study suggest that autologous injection of PRP might be a safe and effective therapeutic tool for the management of delayed union of long bone fractures.

Role of leukotriene B4 (LTB4)-LTB4 receptor 1 signaling in post-incisional nociceptive sensitization and local inflammation in mice.

Leukotriene B4 (LTB4) is a potent lipid mediator involved in the recruitment and activation of neutrophils, which is an important feature of tissue injury and inflammation. The biological effects of LTB4 are primarily mediated through the high-affinity LTB4 receptor, BLT1. Postoperative incisional pain is characterized by persistent acute pain at the site of tissue injury and is associated with local inflammation. Here, we compared the role of LTB4-BLT1 signaling in postoperative incisional pain between BLT1-knockout (BLT1KO) and wild-type (BLT1WT) mice. A planter incision model was developed, and mechanical pain hypersensitivity was determined using the von Frey test before and after incision. Local infiltration of neutrophils and inflammatory monocytes was quantified by flow cytometry. Inflammatory cytokine levels in the incised tissue were also determined. Mechanical pain hypersensitivity was significantly reduced in BLT1KO mice compared to BLT1WT mice at 2, 3, and 4 days after incision. LTB4 levels in the tissue at the incision site peaked 3 hours after the incision. Infiltrated neutrophils peaked 1 day after the incision in both BLT1KO and BLT1WT mice. The accumulation of inflammatory monocytes increased 1-3 days after the incision and was significantly more reduced in BLT1KO mice than in BLT1WT mice. In BLT1KO mice, Interleukin-1β and Tumor Necrosis Factor-α levels 1 day after the incision were significantly lower than those of BLT1WT mice. Our data suggest that LTB4 is produced and activates its receptor BLT1 in the very early phase of tissue injury, and that LTB4-BLT1 signaling exacerbates pain responses by promoting local infiltration of inflammatory monocytes and cytokine production. Thus, LTB4-BLT1 signaling is a potential target for therapeutic intervention of acute and persistent pain induced by tissue injury.

Inflammation Self-Limiting Electrospun Fibrous Tape via Regional Immunity for Deep Soft Tissue Repair.

Overexpression of inflammatory cytokines and chemokines occurs at deep soft tissue injury sites impeding the inflammation self-limiting and impairing the tissue remodeling process. Inspired by the electrostatically extracellular matrix (ECM) binding property of the inflammatory signals, an inflammation self-limiting fibrous tape is designed by covalently modifying the thermosensitive methacrylated gelatin (GelMA) and negatively charged methacrylated heparin (HepMA) hydrogel mixture with proper ratio onto the electrospun fibrous membrane by mild alkali hydrolysis and carboxyl-amino condensation reaction to restore inflammation self-limiting and promote tissue repair via regional immunity regulation. While the GelMA guarantees cell compatibility, the negatively charged HepMA successfully adsorbs the inflammatory cytokines and chemokines by electrostatic interactions and inhibits immune cell migration in vitro. Furthermore, in vivo inflammation self-limiting and regional immunity regulation efficacy is evaluated in a rat abdominal hernia model. Reduced local inflammatory cytokines and chemokines in the early stage and increased angiogenesis and ECM remodeling in the later phase confirm that the tape is an approach to maintain an optimal regional immune activation level after soft tissue injury. Overall, the reported electrospun fibrous tape will find its way into clinical transformation and solve the challenges of deep soft tissue injury.

Serum levels of monocyte chemoattractant protein-1 has no correlation with gender and age in polytrauma

&lt;p class="abstract"&gt;&lt;strong&gt;Background:&lt;/strong&gt; Trauma is one of the commonest causes of death among young people with significant increase in morbidity and mortality. Following injuries to tissues, the body responds in an attempt to cause repair of the damaged tissue. Although its significance depends on the interplay of various factors involving neurohormonal and immune responses. Monocyte chemoattractant protein-1 (MCP-1) is a pro-inflammatory chemoattractant produced by the monocyte and causes the further release of chemokines and cytokines needed at the site of tissue injury. It therefore plays a critical role in tissue healing.&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Methods:&lt;/strong&gt; This was a prospective hospital-based study carried out at University College Hospital (UCH), Ibadan. Polytrauma patients admitted through the accident and emergency department that met a defined criteria were recruited and had their blood samples taken into an endotoxin free test tube at 48±2 hours after trauma. MCP-1 levels in the serum were estimated though the human MCP-1 enzyme-linked immunosorbent assay (ELISA) kit. This process was carried out using the ELISA technique based on the producer’s guide.&lt;strong&gt;&lt;/strong&gt;&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Results:&lt;/strong&gt; 110 patients were recruited for the study. There was no significant difference in gender variations in MCP-1 level (t=-0.935, p=0.351). For the male and female variations in MCP-1 levels, it ranges from 10 to 2841 and 22 to 2687 respectively. Likewise, there was no significant relationship in age variations in MCP-1 values (f=0.959, p&amp;gt;0.05).&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Conclusions:&lt;/strong&gt; This study demonstrated no correlation between serum level of monocyte chemoattractant protein-1 with age and gender in polytraumatized patient according to age and gender.&lt;/p&gt;